The present invention relates to flow cytometers.
Flow cytometers (FCM) are instruments by which properties of single cells or other particles in suspension can be determined. Conventionally, an FCM consists of the following basic components:
i. A liquid flow system by which cells in suspension, which may be loaded with fluorescent dye, are transported in a vertical particle stream and passed singly, one after another, across a zone of analysis where they are exposed to an intense light beam. This zone may be located in open air or in a glass flow chamber;
ii. A light source and focussing system which directs a light beam (for example a laser beam) sharply focussed into the zone of analysis within the particle stream such that only a single cell will be exposed to the beam;
iii. An optical detection system, by which the scattered or fluorescent light pulses emitted by each cell at the moment when the cell passes across the beam, is collected, selected according to wavelength and converted into electronic pulses;
iv. An electronic analysis unit by which these pulses are processed and analyzed for the desired information about the cell characteristics which can be obtained from the light pulses.
A conventional optical detection system is shown schematically in FIG. 1, which is a horizontal section through a flow chamber of an FCM.
In FIG. 1, the flow chamber through which the particle stream passes is shown at 2, the section being taken at the point at which the incident light beam intersects the stream. The cell instantaneously exposed to the beam is shown at 4 and the incident light beam is shown at 6. The light pulses which are emitted from the cell 4 are collected perpendicularly to the incident beam 6 within a solid angle (.alpha.) by a lens 8, then passed through a first beam splitter 10a. The light deflected by the first beam splitter 10a is passed through a color filter 12 onto a first photomultiplier PM.sub.1 for transformation into electronic signals. The light transmitted through the first beam splitter 10a meets a second beam splitter 10b. The light respectively deflected and transmitted by the second beam splitter passes through further color filters 14, 16 to further photomultipliers, PM.sub.2 and PM.sub.3. Thus the light pulses are analyzed in three different parts of the wavelength spectrum.
This conventional detection system is disadvantageous in that each part of this system needs to be adjusted for correct location in three dimensions, and even with very experienced operators, initial adjustments and readjustments during measurement may involve several hours work. With systems effecting more than three color analysis, the use of a highly skilled operator is required for operation.
Further, with this conventional system, all analysis is restricted to the two dimensional plane in which the optical system is mounted. An analysis which could be carried on without such restriction would yield more information concerning the light scatter characteristics of cells, and a higher proportion of the omnidirectional, but normally weak, fluorescent light could be collected.